Protein phosphorylation-dephosphorylation represents one of nature's most fundamentally important and widely used mechanisms for the regulation of protein function and, consequently, cellular events. Protein-serine/threonine phosphatase-1 (PP1) represents one of the two most quantitatively important protein-serine/threonine phosphatases in mammalian cells. PP1 is regulated via the reversible binding of two endogenous inhibitors, I-1 and I-2, and to targeting proteins such as the glycogen-targeting subunit, G, which serve to recruit it to specific locations in the cell. The objectives of this study are to (1) identify the sites on PP1 to which each of these regulatory molecules bind, and (2) apply this knowledge to dissect the functional roles that these regulators play in modulating PP1 action toward the key enzymes of glycogen metabolism -- glycogen synthase and glycogen phosphorylase -- in cultured cells. The first specific aim will be accomplished by constructing chimeric PP1 molecules in which potential regulatory interaction domains are incorporated by genetic engineering onto a unique homolog of PP1, PP1-arch from the archaeon Sulfolobus solfataricus, that is lacks the ability to bind eukaryotically-derived regulatory molecules but is still 29% identical to PP1 from rabbit. These studies will be aided in execution and interpretation by the recently published three-dimensional structure of PP1 from rabbit. Knowledge of the structural features of PP1 responsible for binding to regulatory ligands will then be used to construct, by genetic engineering, altered forms of PP1-rabbit each defective in their ability to bind one of its endogenous regulators. These regulationally-impaired forms will be transfected into hepatoma and myoblast cell lines, and the cells challenged with forskolin, an inducer of glycogenolysis, or insulin, an inducer of glycogen synthesis. The regulatory modalities responsible for controlling PP1 action toward glycogen synthase and glycogen phosphorylase under each circumstance will be identified by virtue of the inability of cells transfected with an altered PP1 to interact with that regulator to trigger a response similar in direction or magnitude to that seen in cells transfected with wild-type PP1. Successful completion of this project may increase the understanding of how PP1 is regulated in cells, and do so in molecular detail.